The long term objectives are as follows: (1) Use cro repressor as a model system for learning how proteins recognize DNA sequences. First we want to determine how cro recognizes its operator. This will be achieved by site specific mutagenesis of cro and selected nucleoside analog substitutions into cro operator. We also want to learn how to change cro at defined positions so that it now recognizes new DNA sequences. Achievement of this goal will aid us in understanding cro repressor-operator interactions and generally how proteins recognize DNA. Finally, how this protein interacts in a nonspecific binding mode with both operator and nonoperator DNA will be studied. (2) Investigate the lac repressor- operator interaction. Recently we discovered that lac repressor binds with high affinity to three sequence divergent operators. Using base analogs, we went to probe the mechanism whereby this protein binds with high affinity to several sequences. (3) Investigate whether repressors and operators can be used diagnostically or therapeutically. The possible diagnostic use will depend upon redesigning cro to recognize nonoperator sequences. Success would encourage us to test a sandwich assay for detecting DNA sequences using mutant cro repressors and cro antibodies. Therapeutic uses for gene control sequences will be explored with a model system, the lac operator. We will attempt to generate a nuclease resistant operator and use it in E. coli to derepress chromosomal lac expression. (4) Determine how regions of E. coli promoters contribute to formation of closed and open complexes. The goal is to measure the kinetics of forming open and closed complexes with promoters that are either covalently crosslinked or lacking defined functional groups. These experiments should identify functional groups involved in initial recognition and in forming an open complex. (5) Investigate components of RNA splicing. First we want to purify and functionally characterize a factor we have isolated from HeLa cell nuclear extracts that is required to reconstitute an active hnRNA splicing complex. We also will purify the debranching enzyme from HeLa cells and characterize its enzymatic activity. Currently we are using a synthetic branched tetranucleotide for these experiments. These studies will be extended to other branch structures having different sequences as well as base and phosphorus analogs. Finally, we plan to examine the catalytic activity of small ribozymes by synthetically introducing various analogs and measuring ribozyme activity.
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